Method of Detecting and/or Quantifying Expression of a Target Protein Candidate in a Cell, and a Method of Identifying a Target Protein of a Small Molecule Modulator

ABSTRACT

The present invention relates to a method of detecting and/or quantifying expression of a target protein candidate in a cell, and to a method of identifying a target protein of a small molecule modulator.

The present invention relates to a method of detecting and/orquantifying expression of a target protein candidate in a cell, and to amethod of identifying a target protein of a small molecule modulator.

The molecular basis of many aspects of cell and protein function, inparticular mammalian cell and protein function, remain elusive in manycases, and there is a need for methods that permit the exploitation ofthe genomic tools in the context of cell based, visual screens ofprotein function. This is of benefit for the identification of theprotein networks operating around and within a cell biological system ofinterest and for the identification of the target(s) of small moleculemodulators identified by chemical genetic (Eggert and Mitchison, 2006)and similar high throughput screening approaches. A current bottleneckin cell based high content screens of protein function is theidentification of these targets (Peterson et al., 2006).

Moreover, in many instances, it is even difficult to reliably andquantitatively detect expression of a target protein in a cell. Someapproaches in the prior art have focussed on the use of nucleic acidarrays, and the effect on the influence of external agents or pathogenicstates on the expression profiles of nucleic acids within the array.This approach, in many instances, is merely qualitative at its best.Moreover, the output of such nucleic acid array experiments generates atremendous amount of data the major part of which may be not directlyamenable to further analysis. Moreover, this type of experiment onlyprovide an insight into the expression of nucleic acids, and anyinference with regard to protein expression is only indirect.Accordingly, there exists a need in the art to provide for a method ofdetecting and/or quantifying expression of a protein in a cell, whichmethod is easy to perform and to analyze.

Small molecules are valuable tools to study dynamic biological processeswith high temporal resolution. Small molecules may also catalyzetherapeutic drug discovery. Pharmaceutically active small molecules maybe discovered by screening chemical libraries for alteration of aspecific protein activity in pure protein in-vitro-assays or for adesired phenotype in cell-based assays. In the past, cell-based assayshave usually been established to report on effects on the activity of asingle pathway or process, typically using a luminescence orfluorescence signal as readout measured in a plate reader. Incell-imaging assays, fields of cells are visualized using fluorescenttags attached to different macromolecules, and automated microscopy.Such data potentially contain a large amount of information relevant todesired as well as unexpected phenotypic changes, and the approach issometimes called “high content screening”. The potential of this methodin academia and industry for the discovery and characterization ofuseful compounds is high, but many challenges remain, particularly atthe level of target identification and data analysis.

Small molecule imaging screens typically involve a number of steps.First of all, usually cells are plated into optical-bottom-multiwellplates, treated with small molecules and incubated for an appropriateperiod of time. Proteins of interest are then rendered fluorescent bysome combination of small molecule fluorescent probes,immunofluorescence with antibodies, and/or the expression of fluorescentprotein (for example GFP)-tag proteins in cells. The latter method canbe applied to both live and fixed cells. In a second step, fluorescentimages are captured by automated microscopy, whereupon the images arethen analyzed to provide measures of phenotypic change, identifyingwells that contain desired, undesired or unexpected phenotypes. In smallmolecule screening methods, it is eventually necessary to identify thebiochemical target causing the phenotypic change. This can bechallenging, especially if phenotypic change results from perturbationof more than one macromolecule.

Consequently, there exists a need in the prior art to provide forimproved methods of identifying target proteins of small moleculemodulators, which methods are easy to perform and which are amenable tofacile analysis. Hence it was an object of the present invention toprovide for a method that permits target identification, i.e. theidentification of macromolecular targets for small molecule modulators,without the need for complex biochemical assays. Moreover, it has beenan object of the present invention to provide for a method which mayserve as a genome wide approach for target identification which avoidslabor intense biochemical methods such as those that exist in the priorart. A method is described that permits the identification of themacromolecular targets for small molecule modulators through theexpression of a palette of potential target nucleic acids, preferablycDNAs, or the repression of their expression by identifying those genesthat modulate the activity of the small molecule modulator.Specifically, the molecular targets of small molecules are identifiedthrough their ability to revert a small molecule phenotype or potentiateit. The method permits target identification without the need forcomplex biochemical assays and thus serves as a genome wide approach fortarget identification, in contrast to the labor intensive biochemicalmethods that exist in the prior art (Peterson et al 2006).

The objects of the present invention are solved by a method of detectingand/or quantifying expression of a target protein candidate in a cellcomprising the steps:

-   -   introducing a first nucleic acid encoding a marker protein into        a vector,    -   introducing a second nucleic acid encoding said target protein        candidate the expression of which is to be detected and/or        quantified, into said vector, such that said first and second        nucleic acids are operably linked, such that expression of said        marker protein is an indication of expression of said target        protein candidate,    -   introducing said vector into a cell,    -   detecting and/or quantifying expression of said marker protein,    -   relating said expression of said marker protein to expression of        said target protein candidate, and thereby detecting and/or        quantifying expression of said target protein candidate.

In one embodiment said first and second nucleic acids are operablylinked within said vector by one of the following arrangements:

a) said first nucleic acid is under control of a first promoter, andsaid second nucleic acid is under control of a second promoter that islocated separately from said first promoter, and said first and secondpromoters are identical in sequence or have identical activity,b) said first nucleic acid is under control of a first promoter, andsaid second nucleic acid is under control of a second promoter that islocated separately from said first promoter, and said first and secondpromoters are not identical in sequence, the activities of each of saidpromoters are predictable,c) said first and said second nucleic acids are under control of asingle promoter, and said first and said second nucleic acids areseparated from each other by a stretch of nucleotides containing aninternal ribosome entry site (IRES).

In one embodiment said marker protein is a fluorescent protein, afragment of an antibody, an epitope, an enzyme, monomeric avidin, apeptide biotin mimic, a peptide that can be detected through directbinding, or by a peptide that can be detected through a chemical bindingwith or reaction with an organic molecule containing a chemicalfluorophore or similar structure such that an optically detectablesignal is produced.

In one embodiment said IRES is selected from IRES from viruses, IRESfrom cellular mRNAs, in particular IRES from picornavirus, such aspolio, EMCV and FMDV, flavivirus, such as hepatitis C virus (HCV),pestivirus, such as classical swine fever virus (CSFV), retrovirus, suchas murine leukaemia virus (MLV), lenlivirus, such as simianimmunodifficiency virus (SIV), and insect RNA virus, such as cricketparalysis virus (CRPV), and IRES from cellular mRNAs, e.g. translationinitiation factors, such as eIF4G, and DAP5, transcription factors, suchas c-Myc, and NF-κB-repressing factor (NRF), growth factors, such asvascular endothelial growth factor (VEGF), fibroblast growth factor 2(FGF-2), platelet-derived growth factor B (PDGF-B), homeotic genes, suchas antennapedia, survival proteins, such as X-linked inhibitor ofapoptosis (XIAP), and Apaf-1, and other cellular mRNA, such as BiP.

In one embodiment

-   -   if said first and second promoters are identical in sequence,        they are selected from the group comprising CMV, EF1, SV40,        human H1 and U6 promoters    -   if said first and second promoters have identical activity, each        of them is independently selected from the group comprising CMV,        EF1, SV40, human H1 and U6 promoters    -   if said first and second promoters are not identical in sequence        and the activities of said promoters are predictable, each of        said promoters is independently selected from the group        comprising CMV, EF1, SV40, human H1 and U6 promoters    -   said single promoter is selected from the group comprising CMV,        EF1, SV40, human H1 and U6 promoters, and said IRES is selected        from the group comprising the nucleic acids having a sequence        selected from the group comprising SEQ ID NO: 1-15. In a        preferred embodiment said single promoter is a CMV promoter and        said IRES is from EMCV. In a particularly preferred embodiment,        said promoter is the CMV immediate early (IE) promoter        (pCMV_(IE)) and the IRES is from EMCV; preferably said IRES from        EMCV is a nucleic acid having a sequence selected from SEQ ID        NO:9, 14 and 15, more preferably 14 and 15 and most preferably        14.

In one embodiment said introducing of said vector into said cell occursby transformation, transfection, electroporation, viral transduction,transduction, ballistic delivery.

Preferably, said detecting and/or quantifying occurs by any opticaldetection method capable of quantitative measurement, in particular, anoptical detection with a spatial resolution, microscopy, fluorescenceactivated cell sorting, UV-Vis spectrometry, fluorescence orphosphorescence measurements, bioluminescence measurements, wherein,more preferably, said microscopy is selected from the group comprisinglight microscopy, bright field microscopy, polarization microscopy,fluorescence microscopy, in particular confocal fluorescence microscopy,evanescent wave excitation microscopy, fluorescence correlationspectroscopy, fluorescence life time microscopy, fluorescence crosscorrelation microscopy, fluorescence recovery after photobleachingmicroscopy, line scanning imaging, point scanning imaging, structuredillumination, deconvolution microscopy, photon counting imaging.

In one embodiment said target protein candidate and said marker proteinare expressed in said cell as separate proteins.

The objects of the present invention are also solved by a method ofidentifying a target protein of a small molecule modulator comprisingthe steps:

-   -   providing a first cell of a type that is capable of producing a        signal when said first cell is exposed to a small molecule        modulator, wherein said signal is a signal that can be spatially        resolved and, optionally, be quantified, preferably by        microscopy,    -   exposing said first cell to a small molecule modulator and        spatially resolving and, optionally, quantifying a first signal        that is produced by said first cell as response to said small        molecule modulator,    -   providing a second cell of the same type as said first cell and    -   performing the method according to any of claims 1-9 on said        second cell,    -   during performance of the method according to any of claims 1-9        on said second cell, after introducing said vector into said        second cell, exposing said second cell to said small molecule        modulator, and spatially resolving and, optionally, quantifying        a second signal that is produced by said second cell as response        to said small molecule modulator,    -   comparing said first signal with said second signal, and, if        there is a difference between said first signal and said second        signal, attributing said difference to the expression of said        target protein candidate in said second cell, thereby        identifying said target protein candidate as a target protein of        said small molecule modulator.

Preferably, said first signal and said second signal are optical signalsthat can be detected, spatially resolved, and, optionally, quantified,by microscopy, wherein, more preferably, said first signal and saidsecond signal are fluorescence signals.

In one embodiment the expression of said marker protein produces a thirdsignal that can be spatially resolved and distinguished from said firstand second signals, wherein, preferably said third signal can bedetected, spatially resolved and, optionally, quantified, by microscopy.

In a preferred embodiment said third signal is a fluorescence signal,and said first and second signals are fluorescent signals, and saidthird signal is spectrally distinct from said first and second signals.

In one embodiment said first signal and said second signal can only bedistinguished from each other by their respective quantity.

Preferably, the method according to the present invention, for a givensmall molecule modulator, is performed with more than one, preferably aplurality of target protein candidates, wherein, more preferably, for agiven small molecule modulator, it is performed with all possible targetprotein candidates of a genome of an organism.

In one embodiment the method according to the present invention isperformed with more than one, preferably a plurality of small moleculemodulators.

The objects of the present invention are also solved by a method ofidentifying a target protein of a small molecule modulator comprisingthe steps:

-   -   providing a first cell of a type that is capable of producing a        signal when said cell is exposed to a small molecule modulator,        wherein said signal is a signal that can be spatially resolved        and, optionally, be quantified, preferably by microscopy,    -   exposing said first cell to a small molecule modulator and        spatially resolving and, optionally, quantifying a first signal        that is produced by said first cell as response to said small        molecule modulator, performing this step at a number of        different concentrations of said small molecule modulator to        determine a first half maximum active concentration (AC₅₀) of        said small molecule modulator which is the half maximum active        concentration in the absence of an inhibition of expression of        said target protein candidate,    -   determining said first half maximum active concentration,    -   providing a second cell of the same type as said first cell and    -   introducing into said second cell small inhibitory RNA (siRNA)        that is selected so as to inhibit expression of a target protein        candidate in said second cell, preferably transfecting said        second cell using small inhibitory RNA (siRNA) that is selected        so as to inhibit expression of a target protein candidate in        said second cell,    -   exposing said second cell to said small molecule modulator and        spatially resolving and, optionally, quantifying a second signal        that is produced by said second cell as response to said small        molecule modulator, performing this step at a number of        different concentrations of said small molecule modulator to        determine a second half maximum active concentration (AC₅₀) of        said small molecule modulator which is the half maximum active        concentration in the presence of an inhibition of expression of        said target protein candidate,    -   determining said second half maximum active concentration,    -   comparing said first half maximum active concentration with said        second half maximum active concentration, and, if there is a        difference between said first half maximum active concentration        and said second half maximum active concentration, attributing        said difference to the inhibition of expression of said target        protein candidate, thereby identifying said target protein        candidate as a target protein of said small molecule modulator.

Preferably, said first and second half maximum active concentrations arehalf maximum inhibitory concentrations (IC₅₀), and said small moleculemodulator is an inhibitor.

In another embodiment said first and second half maximum activeconcentrations are half maximum enhancing concentrations (EC₅₀) and saidsmall molecule modulator is an enhancer.

It should be noted that the term “half maximum active concentration(AC₅₀)”, as used herein, in some embodiments may also refer to anotherproportion of activity. For example, it may refer to a “90% maximumactive concentration (AC₉₀)” or another percentage value that can beused as a discriminatory value, such as 60%, 70% or 80% etc. (AC₆₀,AC₇₀, AC₈₀ etc.). Again, in this case, this percentage then applies alsoto the terms “IC_(x)” and “EC_(x)”, wherein “x” denotes theaforementioned percentage of the maximum active concentration, andwherein “IC” signifies that the small molecule modulator in this case isan inhibitor, and “EC” signifies that the small molecule modulator is anenhancer. In a preferred embodiment, the “half maximum activeconcentration” is a concentration with an activity equal to or greaterthan 50% of the maximum value, i.e. “AC_(≧50)”.

Gene silencing is well known to someone skilled in the art and can beachieved by a plurality of methods (reviewed in Echeverri & PerrimonNature, Reviews Genetics 2006).

Preferably, said first signal and said second signal are optical signalsthat can be detected, spatially resolved, and, optionally quantified, bymicroscopy.

More preferably, said first signal and said second signal arefluorescence signals.

The inventors have managed to devise a method that permits theidentification of the macromolecular targets for small moleculemodulators through the expression of a palette of potential targetcDNAs, or the repression of their expression by identifying those genesthat modulate the activity of the small molecule modulator.Specifically, the molecular targets of small molecules are identifiedthrough their ability to revert a small molecule phenotype or potentiateit. The method permits target identification without the need forcomplex biochemical assays and thus serves as a genome wide approach fortarget identification, in contrast to the labor intensive biochemicalmethods that exist in the prior art (Peterson et al 2006).

In particular, in the embodiment wherein said first and said secondnucleic acids are under control of a single promoter, and said first andsaid second nucleic acids are separated from each other by a stretch ofnucleotides containing an internal ribosome entry site (IRES), thepresent inventors have managed to devise a system particularly usefulfor high-content-screening, because the expression level of said firstnucleic acid matches the expression level of said second nucleic acid.The term “expression level of a nucleic acid”, as used herein, is meantto refer to the expression level of an mRNA or a protein correspondingto said nucleic acid. In this “IRES-embodiment”, there is a1:1-relationship between the expressions and expression levels from eachof said first and said second nucleic acids. In contrast thereto, otherapproaches involving the use of two different promoters may beassociated with a number of problems. For example, promoter interferencephenomena may occur, and there is no guarantee of a reliableco-expression of both nucleic acids. Therefore, such a “dual promoterapproach”, when used in a method identifying a target protein of a smallmolecule modulator, although being encompassed by the present invention,is less preferred, in comparison to an approach where the two nucleicacids are under control of a single promoter and are separated from eachother by a stretch of nucleotides containing an internal ribosome entrysite. In the dual promoter approach for example, the presence of aresistance gene on a transcription unit distinct from the transcriptionunit that expresses the gene of interest, cannot ensure that the latterwill indeed be expressed by resistant cells. For example, if the gene ofinterest has an inhibitory effect on cell proliferation, or it has acell-toxic effect, the selection pressure on a separate transcriptionunit is not able to prevent the counter-selection of the gene ofinterest which therefore leads to resistant clones which only expressthe resistance gene. The “IRES-approach” does not have any of theseproblems, and consequently, this is one of the advantages of thisparticular embodiment.

Furthermore, with respect to another aspect of the present inventioninvolving siRNA, the inventors have devised a method in which a targetprotein of a small molecule modulator is identified by measuring changesin the half-maximum active concentration (AC₅₀) in target genesilencing. In all prior art methods known to the inventors of highcontent screening or high throughput screenings, no such changes in AC₅₀were measured but, if at all, only single point concentrations. If suchsingle point concentration measurements are done at a high concentrationlevel, this usually results in a high rate of false positives, i.e. suchmethod is not at all target-specific. Likewise, some small moleculemodulators may be used well above their “proper” AC₅₀-concentration, inwhich case toxicity effects may be observed and no detection of a targetmight be possible. Furthermore, sometimes with such singleconcentration-measurements, the concentrations effectively used may betoo low, in which case no effect may be detected and therefore targetsmay evade their identification. The present inventors have devised amethod wherein, by performing AC₅₀ titration experiments, they combinethe shift of AC₅₀ with titratable changes in phenotype. Only the propertarget shifts the AC₅₀ and can thereby be distinguished from other geneswhich, in single point concentration measurements, might have possiblyand incorrectly shown up as a target too. Hence, by performingAC₅₀-measurements in a method of identifying a target protein of a smallmolecule modulator using siRNA, the present inventors have devised aparticularly reliable method of high-content-screening.

As used herein, the term “target protein candidate” is meant to refer toany protein, the expression of which one desires to detect and/orquantify. Preferably, such protein is subsequently suspected of being atarget for the action of a small molecule compound. The terms “smallmolecule compound” and “small molecule modulator” are used synonymouslyand interchangeably. A “target protein candidate”, as used herein, issuspected of being a target for a small molecule modulator, but suchsuspicion has not yet been verified. If a “target protein candidate” isidentified as an actual target for a small molecule modulator, such“target protein candidate” is a “target protein” of the small moleculemodulator examined.

The term “operably linked”, as used herein, is meant to refer to anarrangement of two coding nucleic acids with respect to each other, suchthat the expression of the respective proteins coded by these nucleicacids is in a proportional relationship to each other. Hence from thedetection and/or quantification of the expression of one protein encodedby one nucleic acid, one may deduce the presence and/or quantity ofexpression of the second protein coded by the other nucleic acid. In itssimplest form, such proportional relationship may be a linearrelationship, but the invention is not necessarily limited thereto.

An “internal ribosome entry site” or “IRES” as used herein, is meant torefer to a stretch of nucleotides within a coding sequence that allowsfor translation initiation within such coding sequence at the level ofthe mRNA. The protein resulting from such IRES-initiated translation isdifferent to the protein in whose mRNA sequence it is located. Usually,in eukaryotes translation can only be initiated at the 5′-end of themRNA molecule, since 5′ cap recognition is required for the assembly ofthe initiation complex. It is currently believed that an IRES sitemimics the 5′ cap structure. Such IRES sequences were first discoveredin RNA viruses, but have subsequently been identified in a number oforganisms. IRES sites are located in the 5′ untranslated region of RNAand allow the translation of the RNA in a cap-independent manner. Anumber of IRES sites have been identified, and they are characterized bytheir aforementioned ability to initiate translation within an mRNA in acap-independent manner. IRES sites have been comprehensively reviewed(Martinez-Salas et al., Journal of General Virology, 2001, Vol. 82,973-984, and Jackson, R. J., Translational Control of Gene Expression,2000, Sonenberg et al. Eds., pp. 127-184, Cold Spring Harbor N.Y.Furthermore, there exists a regularly updated database for identifiedIRES sites under http://www.iresite.org.

Particularly preferred IRES sites that are used in the method accordingto the present invention are IRES from viral RNAs, such as picornavirus,flavivirus, pestivirus, retrovirus, lentivirus, and insect RNA virus,and IRES sites from cellular mRNAs, such as from translation initiationfactors, transcription factors, growth factors, homeotic genes, andsurvival proteins. Particularly preferred IRES sites are IRES sites frompicornaviruses and flaviviruses, such as EMCV and HCV. Preferred IRESsites for use in the present invention are IRES sites from Drosophilamelanogaster Antennapedia, homeotic gene Antennapedia and Ultrabitorax,human c-myc Oncogene, human v-myc myelocytomatosis viral relatedOncogene, human myelin transcription factor 2 (MYT2) human apoptoticprotease activating factor 1 (Apaf-1), human Coxsackievirus B2 strain20, Encephalomyocarditis virus (EMCV), Cricket paralysis virus, Bovineviral diarrhea virus 1, Hog Cholera virus (Classical swine fever virus),and Hepatitis GB virus B (GBV-B).

More specifically, an IRES site from EMCV is particularly preferred,wherein, preferably, said IRES from EMCV is a nucleic acid having asequence selected from SE ID NO:9, 14 and 15, more preferably 14 and 15,and most preferably 14.

One idea underlying the present invention is the arrangement of anIRES-site between two nucleic acids coding for two proteins ineukaryotic mRNA molecules as bi-cistronic mRNA. In this case, such anIRES site can drive translation of the downstream protein coding regionindependently of the 5′-cap structure bound to the 5′-end of the mRNAmolecule. In this setup, both proteins are produced in the cell, andtheir expression is unitarily coupled. The first protein located in thefirst cistron is synthesized by the cap-dependent initiation approach,while translation initiation of the second protein is directed by theIRES-site located in the intercistronic spacer region between the twoprotein coding regions.

Processes for introducing a nucleic acid into a cell, such astransformation, transfection, electroporation, viral transduction,transduction and/or ballistic delivery are known to someone skilled inthe art and are e.g. reviewed in Sambrook and Russell, 2006.

A “small molecule modulator”, as used herein, is meant to refer to amolecule which is not a protein and the molecular weight of which doesnot exceed 10,000. In the determination of whether a molecule is a“small molecule modulator”, it may also be helpful to consider theso-called Lipinsky rules. Christopher Lipinsky searched for rules fornegative drug design and formulated a rule of 5 set parameters toexclude compounds that would be unlikely to succeed due to poorabsorption or permeation. Hence, according to Lipinsky's rule a moleculeis not likely to be efficiently acting as a small molecule drug ormodulator if it has:

1. more than 5H-bond donors (usually the sum of NHs and OHs),2. more than 10H-bond acceptors (usually the sum of Ns and Os),3. a molecular weight (MW)>500,4. a calculated log P>, and5. a weak inhibition (<100 nM) (Lipinsky et al., 1997, experimental andcomputational approaches to estimate solubility and permeability in drugdiscovery and development settings. ADV. Drug Delivery REV. 23 (1-3).(P=partition coefficient of a molecule between a hydrophobic andhydrophilic environment. In departure from these Lipinsky rules,however, a “small molecule modulator”, as used herein, may also have amolecular weight that is >500 but does not exceed 10000. Morespecifically, a “small molecule modulator”, as used herein, may however,also refer to an oligopeptide or oligonucleotide the molecular weight ofwhich does not exceed 10,000.

A small molecule is also herein referred to as a “modulator” if suchsmall molecule has an effect on the activity of a protein and/or cellmetabolism and/or phenotype. Experimentally, small molecule modulatorsof proteins are often identified using high-throughput screening methodsapplied to chemical libraries. Such libraries may be developed in eithersolid or solution phase and can consist of natural compounds and theirderivatives or synthetic molecules (Hall et al., 2001, J. Comb. Chem.,3:125-150). Chemical libraries are often generated using combinatorialchemistry whereby both functional groups and molecular skeletons ofprecursor compounds are sequentially altered (Burke et al., 2003,Science, 302:613-618; Schreiber, 2000, Science, 287:1964-1969).High-throughput screening has proven to be useful, especially in theareas of drug discovery, agrochemicals and food research.

A method frequently used in performing the method according to thepresent invention is microscopy. A number of types of microscopy existand are known to someone skilled in the art. The term “microscopy”includes but is not limited to optical microscopy, confocal microscopyand automated microscopy screening methods. Particularly preferred formsof microscopy are fluorescence microscopy, in particular confocalfluorescence microscopy, light microscopy, fluorescence lifetimemicroscopy, fluorescence cross correlation microscopy and polarizationmicroscopy. The limitations of microscopy are given by its resolvingpower which is determined by the resolution. Typically, a resolutionlimit is 300 nm-10 μm, preferably 300 nm-1 μm, depending on theparticular type of microscopy used. In this context, the term “spatiallyresolving a signal”, as used herein, is meant to refer to the resolutionof a signal in space to a limit as low as 300 nm-10 μm, preferably 300nm-1 μm, and is typically defined by the Abbé limit.

In this application, sometimes reference is made to “providing a firstcell of type . . . ” and “providing a second cell of type . . . ”. Itwill be clear to someone skilled in the art that, in each of thesesteps, more than one cell of a particular type may be provided, andhence, the performance of the methods according to the present inventionis not restricted to the use of single cells only. In fact, many of theexperiments that are performed in accordance with the method accordingto the present invention, will be performed on cell cultures in whichthere will be a plurality of cells of a particular type. Hence,“providing a first cell of type . . . ” and “providing a second cell oftype . . . ” may also be substituted with “providing a first group ofcells of type . . . ” and “providing a second group of cells of type . .. ”.

As used herein, the term “siRNA” is meant to refer to a stretch of RNAwhich may be used to silence gene expression and/or to interfere withthe expression of a gene. Hence, siRNA is also sometimes referred to as“small interfering RNA”. Such siRNA may be introduced into cells in anumber of ways, one of which is transfection of the cell with suchsiRNA. In another embodiment, however, such gene silencing may beachieved by introducing it into a cell first and allowing its expressiontherein so that a short hairpin RNA is actually expressed within thecell by an appropriate vector, e.g. a plasmid.

The term “half maximum active concentration” or “AC₅₀” as used herein,is meant to refer to the concentration of a small molecule modulator atwhich it shows its half maximum activity. For example, such smallmolecule modulator may be an inhibitor, in which case the activity isinhibition. Consequently, the half maximum active concentration of suchsmall molecule inhibitor is a “half maximum inhibitory concentration” or“IC₅₀”. If the small molecule modulator is an enhancer, such halfmaximum active concentration is a “half maximum enhancing concentration”or “EC₅₀”.

It should be noted that the term “half maximum active concentration(AC₅₀)”, as used herein, in some embodiments may also refer to anotherproportion of activity. For example, it may refer to a “90% maximumactive concentration (AC₉₀)” or another percentage value that can beused as a discriminatory value, such as 60%, 70% or 80% etc. (AC₆₀,AC₇₀, AC₈₀ etc.). Again, in this case, this percentage then applies alsoto the terms “IC_(x)” and “EC_(x)”, wherein “x” denotes theaforementioned percentage of the maximum active concentration, andwherein “IC” signifies that the small molecule modulator in this case isan inhibitor, and “EC” signifies that the small molecule modulator is anenhancer. In a preferred embodiment, the “half maximum activeconcentration” is a concentration with an activity equal to or greaterthan 50% of the maximum value, i.e. “AC_(≧50)”.

The present inventors have found that it is possible to identify atarget protein of a small molecule modulator by examining the impact ofprotein expression on a cell-based assay, wherein the cell-based assayis based on the exposure of cells to a small molecule modulator, as aresult of which exposure a signal is produced by the cell. The outcomeof such cell-based assay is measured in dependence on the extent ofexpression (or absence of expression) of a target protein candidate. Ifthe expression or absence of expression of a target protein candidatehas a detectable influence on the cell-based assay's results, the targetprotein candidate whose expression is artificially induced or increasedor silenced, is a target for the small molecule modulator used in thecell-based assay.

In the following, reference is made to the figures, wherein

FIG. 1 shows confocal laser scanning microscopy photographs of cellswhere green fluorescent protein and red fluorescent protein wereoperably linked by an IRES site, and the expression of either of themcould be used as a measure of expression of the second insert in theIRES vector; panel A (top left corner) shows confocal images of GFPfluorescence in transfected cells, panel B (top right corner) confocalimages of RFP fluorescence in the same cells and panel C (bottom leftcorner) shows a ratio image of the images in A and B representing aarbitrary pseudocolored scale for the GFP:RFP ratio. The correlationbetween GFP and RFP intensity per pixel is shown in panel D (bottomright corner) with GFP intensity on the X axis plotted against RFPfluorescence. The correlation between the intensities in D is0.92+−0.023 (n=5) as described herein,

FIG. 2 shows the effect of transfection and expression of threeIRES-vectors into the endothelin A-receptor-GFP cell line (GFP=greenfluorescent protein); “etar” is the stable cell line transfected with anendothelin A-receptor:IRES:RFP-vector (RFP=red fluorescent protein),“etbr” is the same cell line but transfected instead with an endothelinB-receptor:IRES:RFP-vector, and “kOPr” is the same cell line buttransfected with a kappa-opioid-receptor:IRES:RFP-vector (FIG. 2 a);

FIG. 2B shows the same type of experiment, but this time formed with akappa-opioid-receptor-GFP-cell line;

FIG. 3 shows the result of transfection of three IRES-vectors each ofwhich had a different G-alpha-protein component in it, G-alpha s,G-alpha i and G-alpha q (Gs, Gi and Gq).

FIG. 4 shows the use of the IRES approach for the identification oftarget proteins;

FIG. 4A shows the result of the effect of compound Gö6976 on the agonistinduced internalization of the endothelin A-receptor;(DMSO=dimethylsulfoxide, ET-1=endothelin-1);

FIG. 4B shows the results of transfection experiments of endothelinA-receptor-GFP-cells with IRES:RFP-constructs bearing either proteinkinase c-alpha or protein kinase c-beta;

FIG. 4 c shows the same results as FIG. 4B, but this time with thenumbers of endosomes being normalized to the control at eachconcentration (Go=Gö 6976);

FIG. 5 shows the quantitation of NF-Kappa-B nucleo-cytoplasmic transportin a high throughput immunofluorescence assay;

FIG. 6 shows the results of a parallel experiment, where cells weretransfected with a specific siRNA or a unspecific scrambled siRNA, andleptomycin sensitive NF Kappa-B transport was measured,

FIG. 7 shows a schematic representation of the construct that was usedin the experiments with red fluorescent protein and green fluorescentprotein in example 1,

FIG. 8 shows an example of a commercially available vector that may beused for bicistronic expression of two protein inserts linked by an IRESsite. For example the commercially available vectorpIRES2-DsRed-Express-Vector commercially available from ClontechLaboratories, Inc., USA, may be used. Alternatively, theDsRed-Express-Gene i.e. the red fluorescent protein may be replaced byany other gene of interest, if upstream of the IRES ameasurable/detectable protein is inserted, such as GFP or RFP. There arecommercially available vectors to that extent which have multiplecloning sites upstream and downstream of the IRES site, such as thepIRES-vector of Clontech Laboratories, Inc., USA.

FIG. 9 shows the effect of siRNA on the IC₅₀ of Leptomycin B on thenuclear import of NfκB when alternative SiRNAs that are mechanisticallyrelevant are silenced under the same conditions as in FIG. 6.

Moreover, reference is made to the following sequences, wherein SEQ IDNO:1 is the IRES from NM_(—)206445 Drosophila melanogaster AntennapediaCG1028-RJ, transcript variantJ (Antp), mRNA. IRES name: Antp-D(1323-1574, 252 bp) as referenced in Oh S. K., Scott M. P., Sarnow P.(1992) Homeotic gene Antennapedia mRNA contains 5′-noncoding sequencesthat confer translational initiation by internal ribosome binding.Genes. Dev. 6 (9): 1643-1653.

SEQ ID NO:2 is the IRES, name: Antp-DE (1323-1730, 408 bp) as referencedin Oh S. K., Scott M. P., Sarnow P. (1992) Homeotic gene AntennapediamRNA contains 5′-noncoding sequences that confer translationalinitiation by internal ribosome binding. Genes. Dev. 6 (9): 1643-1653,and Ye X., Fong P., Iizuka N., Choate D., Cavener D. R. (1997)Ultrabithorax and Antennapedia 5′ untranslated regions promotedevelopmentally regulated internal translation initiation. Mol. Cell.Biol. 17 (3): 1714-1721,

SEQ ID NO:3 is the IRES, name: Antp-CDE (1-1730, 1730 bp) as referencedin Oh S. K., Scott M. P., Sarnow P. (1992) Homeotic gene AntennapediamRNA contains 5′-noncoding sequences that confer translationalinitiation by internal ribosome binding. Genes. Dev. 6 (9): 1643-1653,and Ye X., Fong P., Iizuka N., Choate D., Cavener D. R. (1997)Ultrabithorax and Antennapedia 5′ untranslated regions promotedevelopmentally regulated internal translation initiation. Mol. Cell.Biol. 17 (3): 1714-1721,

SEQ ID NO:4 is the IRES from V00568 Human mRNA encoding the c-myconcogene. IRES name: c-myc (1-395, 395 bp) as referenced in Stoneley M.,Paulin F. E., Le Quesne J. P., Chappell S. A., Willis A. E. (1998) C-Myc5′ untranslated region contains an internal ribosome entry segment.Oncogene. 16 (3):423-428,

SEQ ID NO: 5 is the IRES from NM_(—)005378 Homo sapiens v-mycmyelocytomatosis viral related oncogene, neuroblastoma derived (avian)(MYCN), mRNA.

IRES name: N-myc (989-1308, 319 bp) as referenced in

Jopling C. L., Willis A. E. (2001) N-myc translation is initiated via aninternal ribosome entry segment that displays enhanced activity inneuronal cells. Oncogene. 20 (21):2664-2670,

SEQ ID NO: 6 is the IRES from AF006822 Homo sapiens myelin transcriptionfactor 2 (MYT2) mRNA, complete cds

IRES name: MYT2_(—)997-1152 (997-1152, 156 bp) as referenced in

Kim J. G., Armstrong R. C., Berndt J. A., Kim N. W., Hudson L. D. (1998)A secreted DNA-binding protein that is translated through an internalribosome entry site (IRES) and distributed in a discrete pattern in thecentral nervous system. Mol. Cell. Neurosci. 12 (3): 119-140,

SEQ ID NO:7 is the IRES from AF013263 Homo sapiens apoptotic proteaseactivating factor 1 (Apaf-1) mRNA, complete cds. IRES name: Apaf-1(345-577, 233 bp) as referenced in Coldwell M. J., Mitchell S. A.,Stoneley M., MacFarlane M., Willis A. E. (2000) Initiation of Apaf-1translation by internal ribosome entry. Oncogene. 19 (7):899-905,

SEQ ID NO: 8 is the IRES from AY752946 Human coxsackievirus B3 strain20, complete genome. IRES name: CVB3 (1-750, 750 bp) as referenced inJang G. M., Leong L. E., Hoang L. T., Wang P. H., Gutman G. A., SemlerB. L. (2004) Structurally distinct elements mediate internal ribosomeentry within the 5′-noncoding region of a voltage-gated potassiumchannel mRNA. J. Biol. Chem. 279 (46):47419-47430, and

Jimenez J., Jang G. M, Semler B. L., Waterman M. L. (2005) An internalribosome entry site mediates translation of lymphoid enhancer factor-1.RNA. 11 (9): 1385-1399,

SEQ ID NO: 9 is the IRES from NC_(—)001479 Encephalomyocarditis virus,complete genome. IRES name: EMCV (257-832, 576 bp) as referenced in WangZ., Weaver M., Magnuson N. S. (2005) Cryptic promoter activity in theDNA sequence corresponding to the pim-1 5′-UTR. Nucleic Acids Res. 33(7):2248-2258,

SEQ ID NO: 10 is the IRES from NC_(—)003924 Cricket paralysis virus,complete genome IRES name: CrPV_(—)5NCR (1-708, 708 bp) as referenced inWilson J. E., Powell M. J., Hoover S. E., Sarnow P. (2000) Naturallyoccurring dicistronic cricket paralysis virus RNA is regulated by twointernal ribosome entry sites. Mol. Cell. Biol. 20 (14):4990-4999,

SEQ ID NO: 11 is the IRES from NC_(—)001461 Bovine viral diarrhea virus1, complete genome IRES name: BVDV1_(—)1-385 (1-385, 385 bp) as cited inChon S. K., Perez D. R., Donis R. O. (1998) Genetic analysis of theinternal ribosome entry segment of bovine viral diarrhea virus.Virology. 251 (2):370-382,

SEQ ID NO: 12 is the IRES from Z46258 Hog cholera virus (Classical swinefever virus) ‘Chinese’ strain (C-strain; EP 0 351 901 B1) encodingpolyprotein. IRES name: CSFV (1-373, 373 bp) as referenced in RijnbrandR., Bredenbeek P. J., Haasnoot P. C, Kieft J. S., Spaan W. J., Lemon S.M. (2001) The influence of downstream protein-coding sequence oninternal ribosome entry on hepatitis C virus and other flavivirus RNAs.RNA. 7 (4):585-597,

SEQ ID NO: 13 is the IRES from NC_(—)001655 Hepatitis GB virus B,complete genome RES name: GBV-B (1023-1467, 445 bp) as referenced inRijnbrand R., Abell G., Lemon S. M. (2000) Mutational analysis of the GBvirus B internal ribosome entry site. J. Virol. 74 (2):773-783.

SEQ ID NO: 14 is the IRES from EMCV as used in the following examples,and

SEQ ID NO: 15 is the IRES from EMCV as published in ww.iresite.org.

Moreover, reference is made to the following examples which are given toillustrate, not to limit the present invention.

EXAMPLES Example 1 Cell Based Assay of Protein Function andQuantification of Protein Expression Agonist Induced ReceptorInternalisation

A cell based assay of protein function—such as agonist induced G-proteincoupled receptor internalization—is devised and applied such that thedesired activity of the protein can be measured or visualized andmeasured in a high content (visual) assay. Typically, for agonistinduced receptor internalization, this may comprise the visualidentification of cells where the receptor is internalized as part of apopulation of cells. This can be quantified by computer aided imagerecognition, for example. The assay can be using fusion proteins to thereceptor that can be expressed in cells and directly visualized in amicroscope, such as the green fluorescent protein (GFP). It can also bean assay where the endogenous cellular receptor is visualized byindirect methods such as immuno-labelling or using a fluorescentagonist. The assay cell line is typically stably transfected. In oneembodiment, the receptor assay is visualized using 488 nM excitation andsuitable emission filters.

The concept presents the following approach to examining the impact ofprotein expression (Target) on the assay. The target is expressed with atranscriptionally related reporter that is spectrally distinct from thatused in the assay. This is designed such that there is a close to linearcorrelation between target expression and reporter expression. Methodsto achieve this include the use of internal ribosomal entry sites, twoidentical activity promoters driving the target and reporter or twopromoters where the activity is predictable. In the simplest case, theexpression of the reporter is a measure/expression ruler to measure theexpression of the target. Here, the present inventors describe the useof internal ribosomal entry sites.

The experiment is performed as follows: a target: :reporter construct istransiently transfected into the assay cells in accordance with standardprotocols (such as cationic lipid:DNA complex transfection, calciumphosphate mediated transfection, polymer based transfection methods,dendrimers as described in Sambrook and Russell, Molecular Cloning 3rdedition chapter 16, CSH press, 2001). These methods are not highlyefficient and so a range of reporter expression is detected. Thisprovides a range of cells expressing the target at measurable levels andthus the impact of expression can be determined by then visualizing theassay in cells expressing the target at different levels. Thus, a commondrawback of cell transfection is used to advantage because of the cellbased resolution of microscopy that permits identification of singlecells. Thus, the assay is measured in cells that were not transfectedand in those transfected and expressing the target at a range ofconcentrations in a single experimental image.

A copy of the green fluorescent protein was inserted into a internalnbosomal entry vector such that the IRES and the red fluorescent protein(dsRED express—a monomeric red fluorescent protein henceforth referredto as RFP) was downstream of it. (FIG. 7) HEK293 cells were transientlytransfected with this vector where expression of the inserts iscontrolled by the pCMV promoter (see also FIG. 8) and then thefluorescent intensities of the green and red fluorescent protein weremeasured by confocal laser scanning microscopy of >25 cells per field ofview using excitation (488/532 nm) and detection optics suited toseparately quantify the expression of the two fluorophores (FIG. 1). Thedetermined correlation coefficient was 0.91 +−0.023 (n=5) between GFPand RFP intensity in confocal images of transfected cells, indicatingthat RFP or GFP expression can be used as a measure of expression of theother respective insert in the IRES vector.

A series of cDNAs were cloned into the IRES:RFP vector encoding a)G-protein coupled receptors, b) small G-proteins or c) protein kinases.the sequence of the IRES used in these and all subsequent experiments isSEQ ID NO: 14 which is an TRES from EMCV. Initially, the IRES vectorswere transiently expressed in cell lines stably expressing a fusionprotein between the endothelin A receptor and a c-terminal copy of thegreen fluorescent protein. Transfection efficiency into this line was onthe order of 50% of cells by monitoring RFP expressing cells aftertransfection with IRES vectors. These vectors comprised either cDNAsencoding the endothelin B receptor, the kappa opioid receptor or theendothelin A receptor 5′ to the IRES and the RFP. The effect ofexpression of the heterologous/external receptors on the agonist inducedinternalization of the endothelin A receptor was then measured. Twohours after cell stimulation with 40 nM endothelin-1, cells were imagedusing an automated confocal microscope. Images of GFP labeled endothelinA receptor stably expressed in the cell lines could then be used todetermine the fraction of cells where receptor endocytosis had occurredand the RFP image was used to determine the degree of endocytosis of theGFP-labelled endothelin A receptor in IRES vector transfected cells inthe same image.

This was performed using both single blind manual counting and automatedimage analysis using custom journals in a commercial image analysispackage (Metamorph offline, Molecular Devices, CA). Cells wereidentified as control (non-RFP-expresser) or expresser (RFP producer),and the expresser cells were further divided into low, medium and highexpresser categories based on the integrated RFP intensity/pixel in theimages. The number of cells showing receptor internalization wasautomatically counted in these four categories and expressed as afraction of the total number of cells in each category.

-   -   a) Three IRES vectors were separately transfected into the        Endothelin A receptor-GFP cell line: the endothelin A        receptor:IRES:RFP, the endothelin B receptor:IRES:RFP, and the        kappa Opioid receptor:IRES:RFP. When compared to control cells        in the same image, expression of the ETAR:IRES:RFP had minimal        effects on ETAR-GFP internalization whereas both the Endothelin        B receptor and the kappa Opioid receptor significantly reduced        ETAR-GFP internalization (FIG. 2A). This indicated that protein        expression per se was a viable method for screening for proteins        involved in a pathway.    -   b) To demonstrate that this method had selectivity across assays        it was repeated using a kappa-Opioid receptor-GFP cell line,        where no significant difference was determined in the assay for        agonist induced opioid receptor internalization for any receptor        expressed using the IRES vectors (FIG. 2B).

To further determine the usefulness of expression, IRES:RFP vectors wereconstructed for expression of three G-alpha protein components ofheterotrimeric G-proteins, Gαs, Gαi, and Gαq. On expression in theETAR-GFP cell line, two G-protein significantly reduced internalization(Gas, Gai) whereas Gq had no significant effect compared to controlcells expressing RFP alone (FIG. 3A).

This demonstrates that the expression analysis strategy functions forreceptors and small signalling proteins.

-   -   c) A series of vectors comprising either the protein kinase C        isoforms, protein kinase C alpha, or protein kinase C beta        inserted 5′ to the IRES:RFP were transfected into the        endothelin-A-receptor-GFP fusion protein stable cells lines. As        shown in FIG. 4, expression of these protein kinase C isoforms        caused no significant change in agonist induced internalization        of the endothelin-A-receptor-fusion protein when compared to        cells transfected with IRES:RFP vectors that possessed no 5′        insert before the IRES and express only RFP.

Example 2 Identification of target proteins for small moleculemodulators

The present inventors determined that the IRES approach could also beused for the identification of target proteins for compounds.

The agonist induced internalization of the endothelin A receptor is >85%blocked when cells are exposed to micro-molar concentrations of thecompound Gö6976 (FIG. 4A) which is a protein kinase C inhibitor(Martiny-Baron et al., 1993).

Protein kinase C has many isoforms and to determine whether the proteinkinase C alpha or protein kinase C beta isoforms could be the targets ofGö6976 in the endothelin A receptor internalization assay, ETAR-GFPcells were transfected with IRES:RFP constructs bearing either proteinkinase C alpha or protein kinase C beta.

Cells were transfected using standard methods (as reviewed in Sambrookand Russell, 2006), In particular lipofection using commerciallyavailable methods and protocols, such as Roche® Fugene6, target systemstargefect and its variance, lipofectamine etc. The cells—comprising bothtransfected and untransfected cells—were then exposed to a increasingconcentration range of Gö6976, the cells were stimulated with theagonist endothelin-1. The number of endosomes per cell—as measure ofEndothelin A receptor activity—was determined by semi-automated imageanalysis in the population of RFP expressing transfected cells comparedto their control untransfected neighbouring cells.

The effect of expression of protein kinase C alpha in PKCα:IRES:RFPtransfected cells was compared to the control untransfected cells in thesame fields of view across the experimental concentration range ofGö6976 using automated confocal microscopy. Typically, cells were firstexposed to a concentration range of Gö6976 for 120 mm in 1% serummedium, then the medium was exchanged for medium containing identicalconcentrations of Gö6976 but supplemented with agonist (ten fold agonistEC50: 40 nM). After incubation, the cells were imaged using an automatedhigh throughput confocal microscope (such as, the Opera from EvotecTechnologies, Hamburg). Images defining the endothelin-A-receptor-GFPdistribution were acquired as were images of the spectrally separate RFPexpresser cells. The two color images per field of cells were aligned tocorrect for optical vignetting, mechanical misalignment (on the order ofan XY displacement of 1-3 μm; Metamorph Offline, Molecular devicescorporation). non-RFP expresser cells were defined as the controlpopulation in the images and RFP expresser cells were segemtnedsegmentedand then seperated into low, medium, and high (lower 20%, median, upper20%) expressers based on the average RFP intensity per cell.

As expected, there was a significant decrease in the number of endosomesper cell in control cells as the concentration of Gö6976 was increased(FIG. 4B left). A similar decrease was observed in cells expressingPKCβ:IRES:RFP (FIG. 4B left), indicating that protein kinase C alpha didnot reverse the effect of the compound Gö6976. In contrast, the effectof expression of protein kinase C beta in PKCβ:IRES:RFP transfectedcells was different. While increasing Gö6976 concentrations reduced thenumber of endosomes in control cells, endosomes were produced inPKCβ:IRES:RFP expressing cells even at the highest contractions ofGö6976 (FIG. 4B). Thus, protein kinase C beta is identified as thetarget of Gö6976 in this assay for it provides ‘gain of function’ andreverses the phenotype and effec of Gö6976.

The identification of protein kinase C beta as the target of Gö6976 isrobustly demonstrated when the numbers of endosomes are normalized tothe control at each concentration (FIG. 4C). As is clear in FIG. 4C,protein kinase C alpha expression and analysis using this system doesnot reverse the effect of Gö6976 while protein kinase C beta expressionreverses the phenotype and is therefore the target of Gö6976 in theassay.

Example 3 Identification of Target Proteins for Small MoleculesModulators Using siRNA Nucleotransport of Transcription Factor NuclearFactor Kappa B

In addition to the method above which comprises gene expression as a‘gain for function’ screen for drug targets, the present inventors alsodemonstrate a method where RNA interference is used to decrease proteinexpression and then determine if this is a target of a drug in questionby a change in the AC50/IC50 concentration of the compound required togive 50% inhibition of the assay. This is a ‘loss of function’ screen.

In the case of nuclear transport as a proof of principle, thetranslocation of the transcription factor Nuclear factor kappa B wasmeasured using high throughput immuno-fluorescent detection of theendogenously expressed NF-κB complex. Briefly, Hela cells were washedtwice with PBS, then fixed for 10 minutes with 4% (w/v) paraformaldehydein PBS, then washed with PBS. Permeabilization was performed with 0.1%TX-100 PBS for 10 minutes, cells were washed in PBS then incubated with1:200 dilution of rabbit anti-NF-KB in 10% Goat serum-PBS overnight at4° C. Plates were washed 3× with PBS for 10 min on an orbital rotator.1:1000 Alexa-488 Goat anti-rabbit secondary antibody was incubated withthe cells for 60 min at room temperature, cells were washed three times10 minutes with PBS on orbital shaker before addition of 5 μM DRAQ5(DRAQ5=nuclear stain) in PBS for 10 minutes at 37° C.

Leptomycin B was used to block nuclear transport and an IC₅₀ of 2 ng/mLor 4.4 nM was derived (FIG. 5). The expression of the nuclear exportprotein exportin 1/CRM1 was reduced by the transfection of smallinhibitory RNAs targeting the exportin 1 sequence and the effect ofleptomycin B on NFkB transport was assessed as above. Exportin 1/CRM 1knock down using siRNA was estimated to be 50% using the silencing ofGFP in a GFP expresser stable cell line in parallel as a bench mark (notshown). The IC50 for leptomycin B in Exportin 1/CRM 1 knock down cellswas significantly reduced—by a factor of >10 fold—as compared to controlcells transfected with a scrambled siRNA with no known homology to humangenes (FIG. 6). Therefore, CRM 1/exportin 1 is identified as the targetfor leptomycin B, as predicted from the literature (Fomerod et al.,1997), establishing that this method of target identification is viable,and would function in the larger context of genome wide screening.

More specifically, FIG. 5 shows Quantitation of NF-κB nucleo-cytoplasmictransport by a high throughput immuno-fluorescence assay, (a)Cytoplasmic NF-κB accumulates in the nucleus of HeLa cells treated with0 (left panel), 1 ng/mL (center panel) or 20 ng/mL leptomycin B for 40min prior to fixation and detection with anti-NF-κB and Alexa 488secondary antibody, and nuclear staining with 10 μM Draq5. Scale bar 20μm. (b) Simulated nuclear localization images with red representing thenucleus, green NF-κB localization where a cytoplasmic distribution ofNF-κB is morphed onto the nucleus in an XZ (upper row) and XY (centerrow) simulated image series from left to right. The bottom row shows thelabeling of cells after 0, 1, 5, 10, 20 ng/mL leptomycin B treatmentwith the green NF-κB labeling overlaid on the nuclear stain from left toright. Scale bar 5 μM. (c) Quantitation of nuclear import on thesimulation images (upper panel) and cell images (lower panel), (d)Determination of the EC₅₀ for leptomycin in terms of nuclearlocalization of NF-κB after incubation with 0-20 ng/mL Leptomycin B,detection of NF-κB and nuclear staining in microtilre plates. The fittedEC50 for leptomycin B was 2.4 ng/mL within 95% confidence interval of1.9 to 3 ng/mL with an R squared of 0.9957 and is representative of >5assays. All images were acquired on an automated confocal.

FIG. 6 shows the quantitation of NF-κB nucleo-cytoplasmic transport by ahigh throughput immuno-fluorescence assay. Cells were transfected witheither crm1 specific or scrambled siRNAs and leptomycin sensitive NFkBtransport was measured after 24 hours. LMB curves were fitted usinggraphpad prism and had an R²>0.95. All images were acquired on anautomated confocal. (LMB=leptomycin B).

Example 4

FIG. 9 shows the effect of siRNA on the IC50 of leptomycin B on thenuclear import of Nf kappa B when alternative siRNAs that aremechanistically irrelevant are silenced under the same conditionsdescribed above in example 3. As in example 3, the translocation of thetranscription factor nuclear factor kappa B was measured using highthroughput immunofluorescent detection of the endogenously expressedNF-kappa B Komplex. Briefly, Hela cells were washed twice with PBS, thenfixed for 10 minutes with 4% (w/v) paraformaldehyde in PBS, then washedwith PBS. Permeabilization was performed with 0.1% TX-100 PBS for 10minutes, cells were washed in PBS then incubated with 1:200 dilution ofrabbit anti-NF-κB in 10% Goat serum-PBS overnight at 4° C. Plates werewashed 3× with PBS for 10 min on an orbital rotator. 1:1000 Alexa-488Goat anti-rabbit secondary antibody was incubated with the cells for 60min at room temperature, cells were washed three times 10 minutes withPBS on orbital shaker before addition of 5 μM DRAQ5 (DRAQ5=nuclearstain) in PBS for 10 minutes at 37° C.

Leptomycin B was used to block nuclear transport, and the IC₅₀ wasmeasured. In the silencing experiments, scrambled siRNAs or siRNAstargeting the green fluorescent protein or protein kinase C isoformswere used. Silencing expression using control scrambled siRNAs, greenfluorescent protein or protein kinase C isoforms is shown.

The data of FIG. 9 demonstrate the specificity that is achievedperforming this experiment. Silencing a series of other cDNAs gives nophenotype and has no effect on the IC₅₀. The experiment of FIG. 9 wasperformed under the same silencing conditions as described in example 3.

REFERENCES

-   Echeverrri C. J. and Perrimon, N. (2006) High throughout RNAi in    cultured cells: a user's guide. Nature reviews genetics 7, 374-384-   Eggert, U. S. and Mitchison, T. J. (2006) Small molecule screening    by imaging. Curr Opin Chem Biol. 2006, 10, 1-6-   Fomerod, M, Ohno, M., Yoshida, M. and Mattaj, I. W. (1997) CRM1 is    an export receptor for leucine-rich nuclear export signals. Cell,    90, 1051-1060.-   Martiny-Baron, G., Kazanietz, M. G., Mischak, H., Blumberg, P. M.,    Kochs, G., Hug, H., Marme, D. and Schachtele, C. (1993) Selective    inhibition of protein kinase C isozymes by the indolocarbazole    Go 6976. J Biol Chem, 268, 9194-9197.-   Peterson, J. R., Lebensohn, A. M., Pelish, H. E. and    Kirschner, M. W. (2006) Biochemical suppression of small-molecule    inhibitors: a strategy to identify inhibitor targets and signaling    pathway components. Chem Biol, 13, 443-452.-   Sambrook and Russell, Molecular Cloning 3rd, edition chapter 16, CSH    press 2001

The features of the present invention disclosed in the specification,the claims and/or in the accompanying drawings, may, both separately,and in any combination thereof, be material for realizing the inventionin various forms thereof.

1. A method of detecting and/or quantifying expression of a targetprotein candidate in a cell comprising the steps: introducing a firstnucleic acid encoding a marker protein into a vector, introducing asecond nucleic acid encoding said target protein candidate theexpression of which is to be detected and/or quantified, into saidvector, such that said first and second nucleic acids are operablylinked, such, that expression of said marker protein is an indication ofexpression of said target protein candidate, introducing said vectorinto a cell, detecting and/or quantifying expression of said markerprotein, and relating said expression of said marker protein toexpression of said target protein candidate, and thereby detectingand/or quantifying expression of said target protein candidate.
 2. Themethod according to claim 1, wherein said first and second nucleic acidsare operably linked within said vector by one of the followingarrangements: a) said first nucleic acid is under control of a firstpromoter, and said second nucleic acid is under control of a secondpromoter that is located separately from said first promoter, and saidfirst and second promoters are identical in sequence or have identicalactivity, b) said first nucleic acid is under control of a firstpromoter, and said second nucleic acid is under control of a secondpromoter that is located separately from said first promoter, and saidfirst and second promoters are not identical in sequence, and theactivities of each of said promoters are predictable, or c) said firstand said second nucleic acids are under control of a single promoter,and said first and said second nucleic acids are separated from eachother by a stretch of nucleotides containing an internal ribosome entrysite (IRES).
 3. The method according to claim 1, wherein said markerprotein is a fluorescent protein, a fragment of an antibody, an epitope,an enzyme, avidin, a peptide biotin mimic, a peptide that can bedetected through direct binding, or by a peptide that can be detectedthrough a chemical binding with or reaction with an organic moleculecontaining a chemical fluorophore or similar structure such that anoptically detectable signal is produced.
 4. The method according toclaim 2, wherein said IRES is selected from IRES from viruses and IRESfrom cellular mRNAs.
 5. The method according to claim 2, wherein, ifsaid first and second promoters are identical in sequence, they areselected from the group consisting of CMV, EF1, SV40, human H1 and U6promoters, if said first and second promoters have identical activity,each of them is independently selected from the group consisting of CMV,EF1, SV40, human H1 and U6 promoters, if said, first and secondpromoters are not identical in sequence and the activities of saidpromoters are predictable, each of said promoters is independentlyselected from the group consisting of CMV, EF1, SV40, human H1 and U6promoters, or said single promoter is selected from the group consistingof CMV, EF1, SV40, human H1 and U6 promoters, and said IRES is selectedfrom nucleic acids having a sequence selected from the group consistingof SEQ ID NO:1-15.
 6. The method according to claim 1, wherein saidintroducing of said vector into said cell occurs by transformation,transfection, electroporation, viral transduction, transduction,ballistic delivery.
 7. The method according to claim 1, wherein saiddetecting and/or quantifying occurs by an optical detection with aspatial resolution, microscopy, fluorescence activated cell sorting,UV-Vis spectrometry, fluorescence or phosphorescence measurements, orbioluminescence measurements.
 8. The method according to claim 7,wherein said microscopy is selected from the group consisting of lightmicroscopy bright field microscopy, polarization microscopy,fluorescence microscopy, confocal fluorescence microscopy, evanescentwave excitation microscopy, fluorescence correlation spectroscopy,fluorescence life time microscopy, fluorescence cross correlationmicroscopy, fluorescence recovery after photo bleaching microscopy, linescanning imaging, point scanning imaging, structured illumination,deconvolution microscopy, and photon counting imaging.
 9. The methodaccording to claim 1, wherein said, target protein candidate and saidmarker protein are expressed in said cell as separate proteins.
 10. Amethod of identifying a target protein of a small molecule modulatorcomprising the steps: providing a first cell of a type that is capableof producing a signal, when said first cell is exposed to a smallmolecule modulator, wherein said signal is a signal, that can bespatially resolved and, optionally, be quantified, preferably bymicroscopy, exposing said first cell to a small molecule modulator andspatially resolving and, optionally, quantifying a first signal that isproduced by said first cell, in response to said small moleculemodulator, providing a second cell of the same type as said first celland performing the method according to claim 1 on said second cell,during performance of the method according to claim 1 on said secondcell, after introducing said vector into said second cell, exposing saidsecond cell to said small molecule modulator, and spatially resolvingand, optionally, quantifying a second signal that is produced by saidsecond cell in response to said small molecule modulator, and comparingsaid first signal with said second signal, and, if there is a differencebetween said first signal and said second signal, attributing saiddifference to the expression of said target protein candidate in saidsecond cell, thereby identifying said target protein candidate as atarget protein of said small molecule modulator.
 11. The methodaccording to claim 10, wherein said first signal and said second signalare optical signals that can be detected, spatially resolved, and,optionally, quantified, by microscopy.
 12. The method according to claim11, wherein said first signal and said second signal are fluorescencesignals.
 13. The method according to claim 10, wherein the expression ofsaid marker protein produces a third signal that can be spatiallyresolved and distinguished from said first and second signals.
 14. Themethod according to claim 13, wherein said third signal can be detected,spatially resolved and, optionally, quantified, by microscopy.
 15. Themethod according to claim 14, wherein said third signal is afluorescence signal, and said first and second signals are fluorescentsignals, and said third signal is spectrally distinct from said firstand second signals.
 16. The method according to claim 11, wherein saidfirst signal and said second signal can only be distinguished from eachother by their respective quantity.
 17. The method according to claim 10which, for a given small molecule modulator, is performed with aplurality of target protein candidates.
 18. The method according toclaim 17, which, for a given small molecule modulator, is performed withall possible target protein candidates of a genome of an organism. 19.The method according to claim 10 which is performed with a plurality ofsmall molecule modulators.
 20. A method of identifying a target proteinof a small molecule modulator comprising the steps: providing a firstcell of a type that is capable of producing a signal when said cell isexposed to a small molecule modulator, wherein said signal is a signalthat can be spatially resolved and, optionally, be quantified,preferably by microscopy, exposing said first cell to a small moleculemodulator and spatially resolving and, optionally, quantifying a firstsignal that is produced by said first cell in response to said smallmolecule modulator, performing this step at a number of differentconcentrations of said small molecule modulator to determine a firsthalf maximum active concentration (AC₅₀) of said small moleculemodulator which is the half maximum active concentration in the absenceof an inhibition of expression of said target protein candidate,determining said first half maximum active concentration, providing asecond cell of the same type as said first cell and introducing intosaid second cell small inhibitory RNA (siRNA) that is selected so as toinhibit expression of a target protein candidate in said second cell,transferring said second cell using small inhibitory RNA (siRNA) that isselected so as to inhibit expression of a target protein candidate insaid second cell, exposing said second cell to said small moleculemodulator and spatially resolving and, optionally, quantifying a secondsignal that is produced by said second cell in response to said smallmolecule modulator, performing this step at a number of differentconcentrations of said small molecule modulator to determine a secondhalf maximum active concentration (AC₅₀) of said small moleculemodulator which is the half maximum active concentration in the presenceof an inhibition of expression of said target protein candidate,determining said second half maximum active concentration, and comparingsaid first half maximum active concentration with said second halfmaximum active concentration, and, if there is a difference between saidfirst half maximum active concentration and said second half maximumactive concentration, attributing said difference to the inhibition, ofexpression of said target protein candidate, thereby identifying saidtarget protein candidate as a target protein of said small moleculemodulator.
 21. The method, according to claim 20, wherein said first andsecond half maximum active concentrations are half maximum inhibitoryconcentrations (IC₅₀), and said small molecule modulator is aninhibitor.
 22. The method according to claim 20, wherein said first andsecond half maximum active concentrations are half maximum enhancingconcentrations (EC₅₀) and said small molecule modulator is an enhancer.23. The method according to claim 20, wherein said first signal and saidsecond signal are optical signals that can be detected, spatiallyresolved, and, optionally, quantified, by microscopy.
 24. The methodaccording to claim 23, wherein said first signal and said second signalare fluorescence signals.